Insulin resistance is a common metabolic complication in aged muscle, and is a primary defect underlying the etiology of type 2 diabetes (T2D). However, the signaling mechanisms in muscle linking age to insulin resistance are unknown. Considering that in 2010, ~27% of individuals 65 yrs and older in the United States were afflicted with T2D, and in the next 20 years the 65 and older population is anticipated to double to ~70 million, it is imperative that this fundamental gap in knowledge be resolved. Thus, the long-term objective of this research is to define the mechanisms underlying muscle insulin resistance in aging. Caloric restriction (CR; defined as 60% of ad libitum [AL] food intake) robustly reverses muscle insulin resistance, and delays its onset during aging. The NAD+-dependent protein deacetylase, sirtuin 1 (SIRT1) is activated by CR, and is thought to be the putative signaling node linking age and CR to muscle insulin action. Surprisingly, however, knowledge regarding the regulatory role of SIRT1 on muscle insulin action, particularly, in vivo, is remarkably limited. Our central hypothesis is that SIRT1 is a key signaling molecule that links aging and CR to muscle insulin action, primarily by modulating the acetylation of major signaling nodes, such as signal transducer and activator of transcription 3 (STAT3), that then influence many signaling pathways, including the insulin signaling pathway. To address this hypothesis, our approach will be to study muscle insulin signaling and sensitivity in response to age and CR using novel mouse models, in which we have manipulated SIRT1 activity specifically in muscle. Our model predicts that changes in SIRT1 activity during aging, and in response to CR, leads to changes in the acetylation status and subsequent activity of SIRT1 target proteins, which directly or indirectly regulates muscle insulin signaling and sensitivity. Specifically, Aim #1 wil elucidate the contribution of SIRT1 and STAT3 to the pathogenesis of muscle insulin resistance in aging mice, whilst Aim #2 will determine whether a SIRT1- STAT3 signaling axis underlies the ability of CR to enhance muscle insulin sensitivity in mice. For these studies, we will measure muscle insulin signaling and insulin sensitivity, in vivo, using hyperinsulinemic- euglycemic clamps, and ex vivo, using 2-deoxyglucose uptake assays, in young (4 months), mid-aged (12 months) and old (20 months) mice fed an AL diet, and compare them to measurements in mice fed a short- term (30 d) or long-term (9 or 17 months) CR diet. Studies will be conducted in 4 different transgenic mice with either a muscle-specific increase of SIRT1 activity, knockout (KO) of SIRT1 deacetylase activity, KO of STAT3 or KO of SIRT1 and STAT3. In Aim #3, we will use mass spectrometry techniques and adenoviral-based studies in muscle cells to identify novel targets of SIRT1 and the functional effects of their acetylation on insulin action. Altogether, these studies will broaden our understanding of the role of SIRT1 in muscle biology, and may have wide-reaching impact on the development of therapies to treat not only muscle insulin resistance, but also other diseases of aging in skeletal muscle.! PUBLIC HEALTH RELEVANCE: Insulin resistance is a common metabolic defect in aged muscle that increases the risk for developing type 2 diabetes. These experiments aim to define why aging muscle becomes insulin resistant, and to define how caloric restriction reverses this insulin resistance, with particular focus on the anti-aging protein, SIRT1. Knowledge gathered by such studies will allow the design of improved therapies to treat insulin resistance.!